本文選題：過氧化氫 + 黏著斑激酶　； 參考：《南方醫(yī)科大學(xué)》2013年碩士論文

【摘要】：背景 晶狀體上皮細(xì)胞(lens epithelial cells, LECs)是位于晶狀體前囊膜下的一層單層立方上皮細(xì)胞,其代謝活躍,具有生長(zhǎng)、分化和創(chuàng)傷刺激后發(fā)生愈合反應(yīng)的能力。晶狀體上皮細(xì)胞起到對(duì)晶狀體的營(yíng)養(yǎng)、代謝、損傷修復(fù)作用,是維持晶狀體透明性的最重要的防線,任何原因(如衰老、營(yíng)養(yǎng)代謝異常、中毒變性、外傷等)破壞LECs的正常結(jié)構(gòu)和功能,都可能導(dǎo)致不同類型的晶狀體混濁。在既往的研究中,人們通過對(duì)比正常人與白內(nèi)障患者的LECs,發(fā)現(xiàn)白內(nèi)障患者LECs有三個(gè)特點(diǎn)：形態(tài)改變、密度下降、局部增殖導(dǎo)致細(xì)胞復(fù)層排列或向后囊移行。這些形態(tài)結(jié)構(gòu)的異常,是晶狀體發(fā)生渾濁的機(jī)制之一。因此,研究晶狀體上皮細(xì)胞的形態(tài)、結(jié)構(gòu)、凋亡等生物學(xué)特性是研究晶狀體疾病的重要基礎(chǔ)。 白內(nèi)障的發(fā)生發(fā)展與晶狀體長(zhǎng)期處于氧化應(yīng)激狀態(tài)有密切的聯(lián)系。通常認(rèn)為,過量的氧化應(yīng)激是外界各種致白內(nèi)障因素作用的共同途徑,它激活一系列的細(xì)胞內(nèi)信號(hào)通路,最終因損傷LECs而導(dǎo)致晶狀體的渾濁。在眾多導(dǎo)致細(xì)胞氧化應(yīng)激的物質(zhì)中,過氧化氫(hydrogen peroxide, H2O2)是最重要的物質(zhì)。正常人的晶狀體及房水中存在一定數(shù)量的自由基,其中H2O2的濃度約為20-30μmol/L,而在白內(nèi)障患者的房水中可高達(dá)660μmol/L,為正�；颊叩�30倍。由于氧化應(yīng)激,LECs膜通透性改變,細(xì)胞內(nèi)的蛋白質(zhì)漏出,房水成分發(fā)生改變,細(xì)胞內(nèi)環(huán)境改變,穩(wěn)定性下降；細(xì)胞內(nèi)蛋白質(zhì)結(jié)構(gòu)發(fā)生改變,細(xì)胞生理功能、透明度受到影響；DNA受損,晶狀體上皮細(xì)胞凋亡,進(jìn)而無(wú)法供給晶狀體代謝所需的營(yíng)養(yǎng)物質(zhì),加重氧化應(yīng)激的損害。這一系列的變化都能導(dǎo)致晶體的渾濁。氧化應(yīng)激激起了白內(nèi)障發(fā)生、發(fā)展過程中惡性循環(huán)�？梢�,H202誘導(dǎo)LECs的氧化損傷是白內(nèi)障發(fā)生發(fā)展的起始途徑,研究晶狀體上皮細(xì)胞的氧化應(yīng)激機(jī)制是研究年齡相關(guān)性白內(nèi)障發(fā)生發(fā)展機(jī)制的重要方面之一。 黏著斑激酶(Focal adhesion kinase, FAK)是一種位于細(xì)胞質(zhì)的非受體型酪氨酸激酶。FAK自發(fā)現(xiàn)起至今已近20年,目前認(rèn)為FAK在誘導(dǎo)細(xì)胞增殖、細(xì)胞粘附、遷移、抗凋亡、纖維化、分化方面都起到了一定的作用。(1)在細(xì)胞增殖方面,細(xì)胞與ECM的連接是細(xì)胞增殖的必要條件。整合素與細(xì)胞外基質(zhì)連接后,在生長(zhǎng)因子的刺激下,FAK被激活,通過MAPK或P13K的激活促進(jìn)細(xì)胞增殖。(2)在細(xì)胞粘附移行方面,已有大量研究證實(shí)FAK與細(xì)胞的移行有重要的關(guān)系,而且細(xì)胞的移行依賴于細(xì)胞外基質(zhì)(extracellular matrixc,ECM)-整合素-FAK這一系列因素的相互作用。(3)在抑制凋亡方面,FAK家族對(duì)細(xì)胞有不同的影響。抑制FAK能導(dǎo)致細(xì)胞的凋亡,這可能與PI3-K/Akt-1和MEK/Erk信號(hào)通路相關(guān)。總而言之,由于FAK位于多條信號(hào)通路的上游,其對(duì)細(xì)胞生物行為的各個(gè)方面都起到一定的調(diào)節(jié)作用。 目的 通過過氧化氫處理晶狀體上皮細(xì)胞,制造氧化應(yīng)激模型,研究氧化應(yīng)激晶狀體上皮細(xì)胞的增殖、移行、凋亡、及形態(tài)學(xué)的改變,同時(shí),觀察細(xì)胞內(nèi)黏著斑激酶的動(dòng)態(tài)表達(dá)及活化程度,初步探討?zhàn)ぶ呒っ甘欠駥?duì)氧化應(yīng)激晶狀體上皮細(xì)胞的調(diào)控功能。 方法： 1、晶狀體上皮細(xì)胞培養(yǎng)與處理：人晶狀體上皮細(xì)胞(HLECs)細(xì)胞株,購(gòu)自美國(guó)ATCC。細(xì)胞用含有10%FBS的低糖DMEM培養(yǎng)基培養(yǎng),于37℃、體積分?jǐn)?shù)5%的CO2飽和濕度的細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng)。細(xì)胞達(dá)到80%-90%融合后,用不含血清、H202含量分別為0,30,50,70,100,300,500,700,1000μmol/L的低糖DMEM細(xì)胞0,30min,3h,6h,12h,24h。 2、CCK-8法檢測(cè)細(xì)胞存活率：將細(xì)胞密度調(diào)至1×108/L,并將細(xì)胞懸液接種于96孔培養(yǎng)板,每孔100μL,置37℃,體積分?jǐn)?shù)5%C02培養(yǎng)箱中孵育,24h細(xì)胞貼壁后棄上清,對(duì)照組(H202濃度為0μmol/L)加100μL低糖DMEM培養(yǎng)液,處理組分別加入H202濃度為30,50,70,100,300,500,700,1000μmol/L的低糖DMEM100μL,每組設(shè)六個(gè)復(fù)孔,繼續(xù)孵育30min,3h,6h,12h,24h。避光取出,棄上清,PBS洗滌兩次,每孔加入100μL培養(yǎng)基和10μL CCK-8,再加入一組空白對(duì)照組(為無(wú)細(xì)胞組,僅加入培養(yǎng)基與CCK-8)至于培養(yǎng)箱內(nèi)2h,全自動(dòng)酶標(biāo)儀進(jìn)行比色,波長(zhǎng)為450nm,測(cè)每孔吸光度A值。計(jì)算藥物對(duì)細(xì)胞的生存率。生存率(%)=(實(shí)驗(yàn)組A值-空白組A值)／(對(duì)照組A值-空白組A值)×100%。 3、細(xì)胞劃痕實(shí)驗(yàn)檢驗(yàn)細(xì)胞移行能力：將進(jìn)入對(duì)數(shù)生長(zhǎng)期的檢測(cè)細(xì)胞用100μL無(wú)菌槍頭在每個(gè)孔中長(zhǎng)滿的單層細(xì)胞上迅速而輕輕地劃1-2道痕,棄培養(yǎng)基,PBS沖洗3遍以去除掉脫落的細(xì)胞及培養(yǎng)基中的細(xì)胞因子。對(duì)照組加入不含H202的培養(yǎng)基,處理組分別加入H202濃度為100,300,500,700,1000μmol/L的低糖DMEM,每組設(shè)4個(gè)復(fù)孔,測(cè)量8個(gè)值,分別于0時(shí),12h,24h拍照觀察,通過圖像處理系統(tǒng)測(cè)量細(xì)胞爬行的距離,比較不同細(xì)胞劃痕修復(fù)速度。 4、流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況：取對(duì)數(shù)生長(zhǎng)期HLECs,接種于6孔板,長(zhǎng)至90%融合后棄上清,移液管吸凈培養(yǎng)液,無(wú)菌PBS液洗細(xì)胞3次。對(duì)照組加入不含H202的培養(yǎng)基,處理組分別加入H202濃度為100μmol/L、1000μmol/L的低糖DMEM處理24h。按照美國(guó)eBioscience公司Annexin V-FITC細(xì)胞凋亡檢測(cè)試劑盒步驟進(jìn)行細(xì)胞凋亡流式細(xì)胞儀檢測(cè)。 5、激光共聚焦顯微鏡觀察細(xì)胞內(nèi)黏著斑激酶的表達(dá)與分布：細(xì)胞爬片后,對(duì)照組加入1000μL低糖DMEM培養(yǎng)液,處理組分別加入H202濃度為100,300,500,700,1000μmol/L的低糖DMEM1000μL置37℃,體積分?jǐn)?shù)5%C02培養(yǎng)箱中孵育24h。棄去上清,PBS清洗三次,4%多聚甲醛室溫固定5min, PBS清洗三次,-80℃甲醇-20℃固定15min, PBS清洗三次。山羊血清封閉1h, PBS清洗后加入1：200兔抗人FAK一抗4℃濕盒中孵育過夜。PBS清洗3次后,Hoechst33258染核1小時(shí),FITC熒光二抗孵育30min后,PBS清洗多余二抗,甘油封片。避光保存,激光共聚焦顯微鏡下觀察。 6、western blot檢測(cè)細(xì)胞內(nèi)FAK和磷酸化FAK的動(dòng)態(tài)表達(dá)：細(xì)胞接種于6孔板上,長(zhǎng)至90%融合,棄上清。PBS清洗后,對(duì)照組加1000μL低糖DMEM培養(yǎng)液,處理組(實(shí)驗(yàn)組)分別加入H2O2濃度為100,300,500,700,1000μmol/L的低糖DMEM1000μL,二氧化碳培養(yǎng)箱內(nèi)培養(yǎng)30min,3h,6h,12h,24h。分別收集細(xì)胞,提取蛋白質(zhì),取15μl蛋白上樣于8%聚丙烯酰胺凝膠進(jìn)行電泳；轉(zhuǎn)移樣品蛋白于PVDF膜上；3%BSA室溫封閉1h,1:1000FAK一抗4℃孵育過夜；TBST洗膜3次,加1：5000的辣根過氧化物酶標(biāo)記的二抗室溫孵育2h; TBST洗膜3次后,浸入增強(qiáng)化學(xué)發(fā)光試劑,暗室X線片壓片曝光,洗片。圖片經(jīng)光密度圖像掃描儀掃描,Flour Chem程序測(cè)定條帶光密度值。 7統(tǒng)計(jì)處理采用SPSS13.0軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。變量采用(x±s)描述。通過One-way ANOVA法分析比較CCK-8各個(gè)時(shí)間點(diǎn)中不同濃度組間的細(xì)胞存活率的差異,比較細(xì)胞劃痕實(shí)驗(yàn)中不同濃度組中細(xì)胞的移行速度的差異,比較流式細(xì)胞結(jié)果中不同濃度處理組間細(xì)胞凋亡、死亡、存活率的差異,比較western blot實(shí)驗(yàn)中不同濃度組間FAK表達(dá)量的差異,LSD法(方差齊)或Dunnelt's T3法(方差不齊)對(duì)組間進(jìn)行兩兩比較。采用Two-way ANOVA分析不同濃度過氧化氫和不同處理時(shí)間對(duì)細(xì)胞移行速度是否存在交互效應(yīng)。P0.05表示有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1、高濃度過氧化氫處理晶狀體細(xì)胞24h后,細(xì)胞形態(tài)發(fā)生改變：貼壁細(xì)胞變得稀疏,細(xì)胞皺縮、輪廓增強(qiáng)、邊緣僵硬,并由原來(lái)的多邊形變成細(xì)長(zhǎng)型,形成偽足,細(xì)胞核與細(xì)胞漿界限不明顯。而FAK分布也集中至細(xì)胞拉長(zhǎng)部位。 2、處理12小時(shí)以內(nèi),500μmol/L濃度以上的過氧化氫對(duì)細(xì)胞有殺傷作用；處理24h時(shí),不同濃度處理組間細(xì)胞存活率差異有統(tǒng)計(jì)學(xué)意義F=17.96,p0.01。30,70,100,300μmol/L組的細(xì)胞較對(duì)照組增殖明顯,其存活率分別達(dá)到1.24±0.03%(p0.01),1.35±0.08%(p0.01),1.75±0.19%(p0.01)與1.37±0.17%(p=0.04),但100μmol/L組與50,70,300μmol/L之間沒有明顯差異(p50=0.20,p70=0.051,p300=0.10)。而500,700,1000μmol/L組細(xì)胞存活率較之對(duì)照組降低(p5000.01,p7000.01,plooo0.010) 3、在無(wú)血清情況下培養(yǎng)細(xì)胞24h,100μmol/L組細(xì)胞移行速度最快,達(dá)到62.23±1.99單位/小時(shí)(p0.01),對(duì)前、后12個(gè)小時(shí)移行速度進(jìn)行兩組間比較(two-way ANOVA)發(fā)現(xiàn),細(xì)胞移行速度除受到H2O2濃度影響外(F=23.34,p0.01),還受到時(shí)間因素的影響(F=27.76,p0.01)。此外,時(shí)間與濃度之間還存在交互效應(yīng)(F=9.61,p0.01)。 4、細(xì)胞凋亡率總體差異有統(tǒng)計(jì)學(xué)意義(F=7.49,p=0.02)。對(duì)組間進(jìn)行兩兩比較發(fā)現(xiàn),100μmol/L濃度處理組細(xì)胞總體凋亡率為2.40±0.01%,低于對(duì)照組的5.04±0.00%(p=0.01)及1000μmol/L濃度組的4.61±0.01%(p=0.02)比。而1000μmol/L濃度組細(xì)胞的凋亡程度與對(duì)照組比差異無(wú)統(tǒng)計(jì)學(xué)意義。 5、0,100,300,500,700及1000μmol/L的H202處理細(xì)胞24h后,細(xì)胞中FAK含量發(fā)生改變。其中,100,300μmol/L濃度組處理24小時(shí)后,FAK表達(dá)量增加,而700,1000濃度處理過后FAK表達(dá)量降低,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。在此過程中,FAK磷酸化被激活,隨處理濃度和處理時(shí)間的改變而改變。 結(jié)論 過氧化氫對(duì)晶狀體上皮細(xì)胞有著雙重影響：1000μmol/L的H202能提高細(xì)胞內(nèi)FAK的表達(dá),促進(jìn)細(xì)胞增殖、移行,抑制細(xì)胞的凋亡。而1000μmol/L的過氧化氫抑制細(xì)胞內(nèi)FAK的表達(dá),對(duì)細(xì)胞生理功能產(chǎn)生抑制作用,并能導(dǎo)致細(xì)胞死亡。
[Abstract]:background
Lens epithelial cells (LECs) is a layer of monolayer cuboid epithelial cells located in the anterior capsule of the lens. Its metabolism is active and has the ability of growth, differentiation and healing after traumatic stimulation. Lens epithelial cells play the role of nutrition, metabolism, repair and repair of the lens, and maintain the transparency of the lens. The most important line of defense, such as aging, abnormal metabolism, toxic degeneration, trauma, etc., destroys the normal structure and function of LECs, which can lead to different types of lens opacities. In the past study, people found three characteristics of LECs in cataract patients by comparing the LECs of normal and cataract patients: morphological changes, The density decreases and the local proliferation leads to the arrangement of cell layers or the migration of the posterior capsule. These abnormalities are one of the mechanisms of turbidity in the lens. Therefore, the study of the morphology, structure and apoptosis of lens epithelial cells is an important basis for the study of lens diseases.
The development of cataract is closely related to the oxidative stress state of the lens for a long time. It is generally believed that excessive oxidative stress is the common way of various external cataract factors. It activates a series of intracellular signaling pathways and eventually causes turbidity of the crystalline body due to the damage of LECs. In the substance, hydrogen peroxide (H2O2) is the most important substance. There are a certain number of free radicals in the lens and aqueous humor of normal people, of which the concentration of H2O2 is about 20-30 mu mol/L, and in the aqueous humor of the cataract patients up to 660 mu, 30 times as high as that of the normal patients. Because of oxidative stress, the permeability of the LECs membrane changes, cells are changed. Protein leakage in the chamber, changes in the composition of aqueous humor, changes in the intracellular environment, and the decline in stability; the changes in the protein structure in the cells, the physiological function of the cells, the transparency of the cells; the damage of DNA, the apoptosis of the lens epithelial cells, which can not supply the nutrients needed for the metabolism of the lens and aggravate the damage of oxidative stress. The changes in the column can cause the turbidity of the crystal. Oxidative stress arouses the occurrence of cataract and the vicious cycle in the process of development. It can be seen that H202 induced oxidative damage of LECs is the beginning of the development of cataract. The study of the oxidative stress mechanism of lens epithelial cells is an important aspect of the study of the mechanism of the development of age related cataract. 1.
Focal adhesion kinase (FAK), a non receptor tyrosine kinase.FAK located in cytoplasm, has been found for nearly 20 years since it has been found. Now, FAK has been considered to play a definite role in inducing cell proliferation, cell adhesion, migration, anti apoptosis, fibrosis and differentiation. (1) in cell proliferation, the connection between cells and ECM is The necessary condition of cell proliferation. After the integrin is connected with the extracellular matrix, FAK is activated by growth factor stimulation, and the activation of MAPK or P13K promotes cell proliferation. (2) there is a large number of studies on cell adhesion and migration that the migration of FAK and cells is important, and the migration of cells depends on the extracellular matrix (extrace Llular matrixc, ECM) - the interaction of integrin -FAK this series of factors. (3) in the inhibition of apoptosis, the FAK family has a different effect on cells. Inhibition of FAK can lead to cell apoptosis, which may be associated with PI3-K/Akt-1 and MEK/Erk signaling pathways. In a word, because FAK is upstream of multiple signal pathways, it is responsible for cellular biological behavior. All aspects play a certain role in regulating.
objective
By treating the lens epithelial cells by hydrogen peroxide, the oxidative stress model was made to study the proliferation, migration, apoptosis and morphological changes of the epithelial cells of the oxidative stress lens epithelial cells. At the same time, the dynamic expression and activation degree of the focal adhesion kinase in the cells were observed, and the modulation of the focal adhesion kinase on the epithelial cells of the oxidative stress lens epithelial cells was preliminarily discussed. Control function.
Method:
1, lens epithelial cell culture and processing: human lens epithelial cell (HLECs) cell line, purchased from American ATCC. cells using a low sugar DMEM medium containing 10%FBS, cultured in a cell culture box with a volume fraction of 5% CO2 saturated humidity. After 80%-90% fusion, the content of H202 content is 0,30,50,70100,30, respectively. 05007001000 - mol/L low sugar DMEM cells 0,30min, 3h, 6h, 12h, 24h.
2, CCK-8 method detected cell survival rate: the cell density was adjusted to 1 x 108/L, and the cell suspension was inoculated to 96 hole culture plate, 100 mu L per pore, 37 centigrade, the volume fraction 5%C02 incubator, the 24h cells after adherence to the supernatant, the control group (H202 concentration was 0 mol/L) and 100 u L low sugar DMEM culture liquid, and the treatment group was added H202 concentration to 30,50,70100 3005007001000 mu mol/L low sugar DMEM100 mu L, each set of six compound holes, continue to incubate 30min, 3h, 6h, 12h, 24h. to take out the light, abandon the supernatant, PBS washing two times, add 100 mu L culture medium and 10 micron L CCK-8, and then add a group of blank control group (for no cell group, only add medium with the culture) as to the incubator Colorimetry, the wavelength was 450nm, and the absorbance of each hole was A. The survival rate of the cell was calculated. The survival rate (%) = (the A value of the experimental group - the blank group A value) / (the A value of the control group - the blank group A value) * 100%.
3, cell scratch test test cell migration ability: to enter the logarithmic growth period of the detection of cells with 100 mu L aseptic gun head in each hole full of the monolayer quickly and gently stroke 1-2 trace, discard medium, PBS rinse for 3 times to remove the cells and cytokines in the culture medium. The control group is added without H202 medium, The treatment group was added to the low sugar DMEM with the concentration of 1003005007001000 H202 mol/L respectively. Each group had 4 compound holes, and 8 values were measured. At 0, 12h and 24h were photographed, and the distance of the cell crawling was measured by the image processing system, and the rate of repair of different cells was compared.
4, flow cytometry detected the cell apoptosis: take the logarithmic growth period HLECs, inoculate the 6 hole plate, long to 90% fusion, remove the supernatant, the pipette suction culture solution, the aseptic PBS liquid washing cell for 3 times. The control group added the medium without H202, and the treatment group was added to the H202 concentration of 100 u mol/L, and the low sugar DMEM processing 24h. of 1000 u mol/L respectively according to American eBiosc Ience Annexin V-FITC apoptosis detection kit was used to detect apoptosis by flow cytometry.
5, the expression and distribution of intracellular sticky kinases were observed by laser confocal microscope: after the cell crawling, the control group was added 1000 L low sugar DMEM culture medium, and the treatment group was added to the low sugar DMEM1000 mu L with the concentration of 1003005007001000 mu mol/L, respectively, and the volume fraction 5%C02 incubator was incubated with 24h. abandoned to the supernatant, PBS cleaning three times, 4% more. Polyoxymethylene was fixed at room temperature for 5min, PBS was cleaned three times, 15min was fixed at -20 C at -80 C -20 C, and PBS was cleaned. The goat serum closed 1H, PBS was cleaned and incubated in the wet box of anti human FAK one anti 4 C for 3 times after cleaning for 1 hours, and after fluorescent two was incubated for 1 hours. It is observed under laser confocal microscope.
6, Western blot detected the dynamic expression of FAK and phosphorylated FAK in cells: the cells were inoculated on the 6 hole plate, long to 90% fusion, and after the cleaning of the supernatant.PBS, the control group was added with 1000 mu L low sugar DMEM culture solution, and the treatment group (experimental group) was added to the H2O2 concentration of 1003005007001000 micron DMEM1000 u L, the incubator of carbon dioxide was incubated for 30min, 3 H, 6h, 12h, 24h., respectively collect cells, extract protein, take 15 mu L protein on 8% polyacrylamide gel electrophoresis; transfer sample protein on PVDF membrane; 3%BSA room temperature closed 1H, 1:1000FAK one anti 4 C incubation for night; TBST washing 3 times, 1:5000 horseradish peroxidase labelled two at room temperature incubating 2H; baptised membrane after 3 times, soak. Enhanced chemiluminescence reagents, darkroom X-ray films, exposures and films. The images were scanned by optical density scanner and the optical density values were measured by Flour Chem program.
7 the statistical analysis of the experimental data was carried out by SPSS13.0 software. The variables were described by (x + s). The difference of cell survival rate between different concentration groups in each time point of CCK-8 was analyzed by One-way ANOVA method, and the difference in the migration velocity of cells in different concentration groups was compared, and the results of flow cytometry were compared. The difference of apoptosis, death and survival rate between different concentration groups was compared, and the difference of FAK expression between different concentration groups in Western blot experiment was compared. The LSD method (Fang Chaqi) or Dunnelt's T3 method (variance uneven) was compared between the 22 groups. The cell migration velocity of different concentration of hydrogen peroxide and different treatment time was analyzed by Two-way ANOVA Whether there was interaction effect,.P0.05 showed statistical significance.
Result
1, after the high concentration of hydrogen peroxide treated the lens cell 24h, the cell morphology changes: the adherent cells become sparse, the cell crinkle, the contour is strengthened, the edge is rigid, and the original polygon becomes slender, forming the pseudo foot, the nucleus and the cytoplasm boundary are not obvious. And the FAK distribution is also concentrated to the elongated part of the cell.
2, under the treatment of 12 hours, the concentration of hydrogen peroxide above 500 mu mol/L had a killing effect on the cells. When treating 24h, the difference of cell survival rate between different concentration treated groups was statistically significant F=17.96, the cells of p0.01.30,70100300 mu mol/L group proliferated significantly compared with the control group, the survival rate was 1.24 + 0.03% (P0.01), 1.35 + 0.08% (P0.01), 1.7 5 + 0.19% (P0.01) and 1.37 + 0.17% (p=0.04), but there was no significant difference between the 100 mu mol/L group and 50,70300 mu mol/L (p50=0.20, p70=0.051, p300=0.10). The survival rate of the 5007001000 micron group was lower than that of the control group (p5000.01, p7000.01, plooo0.010).
3, in serum-free cell culture, cell 24h was cultured, and the speed of cell migration was the fastest, reaching 62.23 + 1.99 units / hours (P0.01). Before and after 12 hours, the rate of migration was compared between the two groups (two-way ANOVA). The rate of cell migration was not affected by H2O2 concentration (F=23.34, P0.01), but also influenced by time factors (F=27.76, P0.01). In addition, there is an interaction effect between time and concentration (F=9.61, P0.01).
4, the overall difference of apoptosis rate was statistically significant (F=7.49, p=0.02). The total apoptosis rate of 100 mu mol/L concentration treatment group was 2.40 + 0.01%, compared with 5.04 + 0% (p=0.01) and 1000 mu mol/L concentration group of 4.61 + 0.01% (p= 0.02) ratio in the control group, and the degree of apoptosis and the control of cells in the 1000 mu mol/L concentration group were compared with those of the control group. There was no significant difference in the group ratio.
After 5,0100300500700 and 1000 mol/L H202 treated cells 24h, the FAK content in the cells changed. Among them, the expression of FAK increased after 24 hours treatment with 100300 mol/L concentration group, and the expression of FAK decreased after 7001000 concentration treatment. The difference was statistically significant (P0.05). In this process, FAK phosphorylation was activated, with treatment concentration and treatment. Change of time.
conclusion
Hydrogen peroxide has a dual effect on lens epithelial cells: 1000 mol/L H202 can increase the expression of FAK in cells, promote cell proliferation, move and inhibit the apoptosis of cells. The 1000 mol/L hydrogen peroxide inhibits the expression of FAK in cells, inhibits the physiological functions of cells and causes cell death.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R776

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